The purpose of this project is to explore the organization of genes encoding components of the NADPH oxidase. This enzyme is responsible for the generation of potent microbicidal oxidants derived from the free radical superoxide anion. The importance of this system in host defense can be appreciated from patients with chronic granulomatous disease (CGD), who suffer from recurrent infections due to genetic lesions in any one of four genes encoding essential protein components of the oxidase. This program will aid in our understanding of the molecular genetic basis for this hereditary immune deficiency and may suggest certain strategies for correction of these gene defects. Current efforts are focused on the gene encoding the cytosolic oxidase factor p67-phox, which was recently mapped to chromosome lq25. In the last year we have isolated and mapped cosmid genomic clones spanning 70 kilobases within this gene locus. This gene is organized into 16 exon segments and corresponding intron boundaries have been located and sequenced. This work has provided insights on the molecular basis for p67-phox deficiency in one autosomal recessive CGD patient, where a deletion at an exon-intron junction predicts a splicing defect that would cause premature termination of protein coding sequence. Other work has defined the location and sequence of a normal restriction fragment length polymorphism (RFLP) within the p67-phox gene, which was used as a genetic marker in prenatal diagnosis of a healthy individual in a family with p67-phox deficient CGD. In addition to obtaining information regarding genetic defects causing CGD, other work has sought to define genetic signals governing myeloid specific gene regulation of p67-phox. A putative gamma-interferon consensus sequence binding protein (ICSBP) binding site was recognized in the genomic sequence upstream from cDNA base -68. Specific gene promoter activity will be studied using reporter gene fusion constructs which contain candidate regulatory sequences. This work may define mechanisms by which NADPH oxidase gene expression may be regulated by gamma-interferon and other immune modulators.